AMPA-binding human GluR1 receptors

ABSTRACT

Described herein are isolated polynucleotides which code for an AMPA-type human CNS receptor, designated the human GluR1B receptor. The receptor is characterized structurally and the construction and use of cell lines expressing the receptor is disclosed.

This application is a continuation, of application Ser. No. 07/896,611,filed Jun. 10, 1992, now abandoned.

FIELD OF THE INVENTION

This invention is concerned with applications of recombinant DNAtechnology in the field of neurobiology. More particularly, theinvention relates to the cloning and expression of DNA coding forexcitatory amino acid (EAA) receptors, especially human EAA receptors.

BACKGROUND OF THE INVENTION

In the mammalian central nervous system (CNS), the transmission of nerveimpluses is controlled by the interaction between a neurotransmittersubstance released by the "sending" neuron which binds to a surfacereceptor on the "receiving" neuron, to cause excitation thereof.L-glutamate is the most abundant neurotransmitter in the CNS, andmediates the major excitatory pathway in vertebrates. Glutamate istherefore referred to as an excitatory amino acid (EAA) and thereceptors which respond to it are variously referred to as glutamatereceptors, or more commonly as EAA receptors.

Using tissues isolated from mammalian brain, and various synthetic EAAreceptor agonists, knowledge of EAA receptor pharmacology has beenrefined somewhat. Members of the EAA receptor family are now groupedinto three main types based on differential binding to such agonists.One type of EAA receptor, which in addition to glutamate also binds theagonist NMDA (N-methyl-D-aspartate), is referred to as the NMDA type ofEAA receptor. Two other glutamate-binding types of EAA receptor, whichdo not bind NMDA, are named according to their preference for bindingwith two other EAA receptor agonists, namely AMPA(alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate.Particularly, receptors which bind glutamate but not NMDA, and whichbind with greater affinity to kainate than to AMPA, are referred to askainate type EAA receptors. Similarly, those EAA receptors which bindglutamate but not NMDA, and which bind AMPA with greater affinity thankainate are referred to as AMPA type EAA receptors.

The glutamate-binding EAA receptor family is of great physiological andmedical importance. Glutamate is involved in many aspects of long-termpotentiation (learning and memory), in the development of synapticplasticity, in epileptic seizures, in neuronal damage caused by ischemiafollowing stroke or other hypoxic events, as well as in other forms ofneurodegenerative processes. However, the development of therapeuticswhich modulate these processes has been very difficult, due to the lackof any homogeneous source of receptor material with which to discoverselectively binding drug molecules, which interact specifically at theinterface of the EAA receptor. The brain derived tissues currently usedto screen candidate drugs are heterogeneous receptor sources, possessingon their surface many receptor types which interfere with studies of theEAA receptor/ligand interface of interest. The search for humantherapeutics is further complicated by the limited availability of braintissue of human origin. It would therefore be desirable to obtain cellsthat are genetically engineered to produce only the receptor ofinterest. With cell lines expressing cloned receptor genes, a substratewhich is homogeneous for the desired receptor is provided, for drugscreening programs.

Very recently, genes encoding substituent polypeptides of EAA receptorsfrom non-human sources, principally rat, have been discovered. Hollmannet al., Nature 342: 643, 1989 described the isolation from rat of a genereferred to originaily as GluR-K1 (but now called simply GluR1). Thisgene encodes a member of the rat EAA receptor family, and was originallysuspected as being of the kainate type. Subsequent studies by Keinanenet al., Science 249: 556, 1990, showed, again in rat, that a gene calledGluR-A, which was in fact identical to the previously isolated GluR1, infact encodes a receptor not of the kainate type, but rather of the AMPAtype. These two groups of researchers have since reported as many asfive related genes isolated from rat sources. Boulter et al., Science249: 1033, 1990, revealed that, in addition to GluR1, the rat contained3 other related genes, which they called GluR2, GluR3, and GluR4, andBettier et al., Neuron 5: 583. 1990 described GluR5. Keinanen et al.,supra, described genes called GluR-A, GluR-B, GluR-C and GluR-D whichcorrespond precisely to GluR1, GluR2, GluR3 and GluR4 respectively.Sommer et al., Science 249: 1580, 1990 also showed, for GluR-A, GluR-B,GluR-C and GluR-D two alternatively spliced forms for each gene. Theseauthors, as well as Monyer et al., Neuron 6: 799, 1991 were able to showthat the differently spliced versions of these genes were differentiallyexpressed in the rat brain. In addition to the isolation of these AMPAreceptor genes, several studies have more recently attempted todetermine the ion-gating properties of different mixtures of the knownreceptors (Nakanishi et al., Neuron 5: 569, 1990; Hollmann et al.,Science 252: 851, 1991; Verdoorn et al., Science 252: 1715, 1991; andsee WO 91/06648).

There has emerged from these molecular cloning advances a betterunderstanding of the structural features of EAA receptors and theirsubunits, as they exist in the rat brain. According to the current modelof EAA receptor structure, each is heteromeric in structure, consistingof individual membrane-anchored subunits, each having four transmembraneregions, and extracellular domains that dictate ligand bindingproperties to some extent and contribute to the ion-gating functionserved by the receptor complex. Keinanen et al, supra, have shown forexample that each subunit of the rat GluR receptor, including thosedesignated GluR-A, GluR-B, GluR-C and GluR-D, display cation channelactivity gated by glutamate, by AMPA and by kainate, in their unitarystate. When expressed in combination however, for example GluR-A incombination with GluR-B, gated ion channels with notably larger currentsare produced by the host mammalian cells.

In the search for therapeutics useful to treat CNS disorders in humans,it is highly desirable of course to provide a screen for candidatecompounds that is more representative of the human situation than ispossible with the rat receptors isolated to date. It is particularlydesirable to provide cloned genes coding for human receptors, and celllines expressing those genes, in order to generate a proper screen forhuman therapeutic compounds. These, accordingly, are objects of thepresent invention.

SUMMARY OF THE INVENTION

The present invention provides an isolated polynucleotide that codes foran AMPA-binding human EAA receptor. By providing polynucleotide thatcodes specifically for a CNS receptor native to humans, the presentinvention provides means for evaluating the human nervous system, andparticularly for assessing potentially therapeutic interactions betweenthe AMPA-binding human EAA receptors and selected natural and syntheticligands.

In one of its aspects, the present invention provides an isolatedpolynucleotide comprising nucleic acids arranged in a sequence thatcodes for a human EAA receptor herein designated the human GluR1Breceptor. Alternatively, the polynucleotide may code for an AMPA-bindingfragment of the human GluR1B receptor, or for an AMPA-binding variant ofthe human GluR1B receptor. In various specific embodiments of thepresent invention, the polynucleotide consists of DNA e.g. cDNA, or ofRNA e.g. messenger RNA. In other embodiments of the present invention,the polynucleotide may be coupled to a reporter molecule, such as aradioactive label, for use in autoradiographic studies of human GluR1Breceptor tissue distribution. In further embodiments of the presentinvention, fragments of the polynucleotides of the invention, includingradiolabelled versions thereof, may be employed either as probes fordetection of glutamate receptor-encoding polynucleotides, as primersappropriate for amplifying such polynucleotides present in a biologicalspecimen, or as templates for expression of the human GluR1B receptor oran AMPA-binding fragment of variant thereof.

According to another aspect of the present invention, there is provideda cellular host having incorporated therein a polynucleotide of thepresent invention. In embodiments of the present invention, thepolynucleotide is a DNA molecule and is incorporated for expression andsecretion in the cellular host, to yield a functional, membrane-boundhuman GluR1B receptor or to yield an AMPA-binding fragment or variant ofthe human GluR1B receptor. In other embodiments of the presentinvention, the polynucleotide is an RNA molecule which is incorporatedin the cellular host to yield the human GluR1B receptor as a functional,membrane-bound product of translation.

According to another aspect of the invention, there is provided aprocess for obtaining a substantially homogeneous source of a human EAAreceptor useful for performing ligand binding assays, which comprisesthe steps of culturing a genetically engineered cellular host of theinvention, and then recovering the cultured cells. Optionally, thecultured cells may be treated to obtain membrane preparations thereof,for use in the ligand binding assays.

According to another aspect of the present invention, there is provideda method for assessing the binding interaction between a test compoundand a human CNS receptor, which comprises the steps of incubating thetest compound under appropriate conditions with a human GluR1B receptorsource, i.e., a cellular host of the invention or a membrane preparationderived therefrom, and then determining the extent or result of bindingbetween the substance and the receptor source.

These and other aspects of the invention are now described in greaterdetail with reference to the accompanying drawings, in which:

BRIEF REFERENCE TO THE DRAWINGS

FIG. 1 provides a DNA sequence (SEQ ID NO:1) coding for the human GluR1Breceptor, and the amino acid sequence (SEQ ID NO:1) thereof;

FIG. 2 depicts the strategy employed in generating recombinant DNAexpression constructs incorporating the human GluR1B receptor-encodingDNA of FIG. 1 (the sequence shown in FIG. 3 are also disclosed in SEQ IDNOs. 3 and 4); and

FIG. 3 illustrates the AMPA-binding property of the human GluR1Breceptor.

DETAILED DESCRIPTION OF THE INVENTION AND ITS PREFERRED EMBODIMENTS

The invention relates to human CNS receptors of the AMPA-binding type,and provides isolated polynucleotides that code for such receptors. Theterm "isolated" is used herein with reference to intact polynucleotidesthat are generally less than about 4,000 nucleotides in length and whichare otherwise isolated from DNA coding for other human proteins.

In the present context, human CNS receptors of the AMPA-binding typeexhibit a characteristic ligand binding profile, which reveals glutamatebinding and relative greater affinity for binding AMPA than for otherbinding other CNS receptor ligands such as kainate, glutamate and theirclosely related analogues.

In the present specification, an AMPA-binding receptor is said to be"functional" if a cellular host producing it exhibits de novo channelactivity when exposed appropriately to AMPA, as determined by theestablished electrophysiological assays described for example by Hollmanet al, supra, or by any other assay appropriate for detectingconductance across a cell membrane.

The human GluR1B receptor of the invention possess structural featurescharacteristic of the EAA receptors in general, including extracellularN- and C-terminal regions, as well as four internal hydrophobic domainswhich serve to anchor the receptor within the cell surface membrane.More specifically, GluR1B receptor is a protein characterizedstructurally as a single polypeptide chain that is produced initially inprecursor form bearing an 18 amino acid residue N-terminal signalpeptide, and is transported to the cell surface in mature form, lackingthe signal peptide and consisting of 888 amino acids arranged in thesequence illustrated, by single letter code, in FIG. 1 and SEQ ID NOs. 1and 2. Unless otherwise stated, the term human GluR1B receptor refers tothe mature form of the receptor, and amino acid residues of the humanGluR1B receptor are accrodingly numbered with reference to the matureprotein sequence. With respect to structural domains of the receptor,hydropathy analysis reveals four putative transmembrane domains, onespanning residues 521-540 inclusive (TM-1), another spanning residues567-585 (TM-2), a third spanning residues 596-614 (TM-3) and the fourthspanning residues 788-808 (TM-4). Based on this assignment, it is likelythat the human GluR1B receptor structure, in its natural membrane-boundform, consists of a 520 amino acid N-terminal extracellular domain,followed by a hydrophobic region containing four transmembrane domainsand an extracellular, 80 amino acid C-terminal domain.

Binding assays performed with various ligands, and with membranepreparations derived from mammalian cells engineered genetically toproduce the human GluR1B receptor in membrane-bound form indicate thatGluR1B binds selectively to AMPA, relative particularly to kainate andNMDA. This feature, coupled with the medically significant connectionbetween AMPA-type receptors and neurological disorders and diseaseindicate that the present receptor, and its AMPA-binding fragments andvariants, will serve as valuable tools in the screening and discovery ofligands useful to modulate in vivo interactions between such receptorsand their natural ligand, glutamate. Thus, a key aspect of the presentinvention resides in the construction of cells that are engineeredgenetically to produce human GluR1B receptor, to serve as a ready andhomogeneous source of receptor for use in in vitro ligand binding and/orchannel activation assays.

For use in the ligand binding assays, it is desirable to construct byapplication of genetic engineering techniques a mammalian cell thatproduces a human GluR1B receptor as a heterologous, membrane-boundproduct. According to one embodiment of the invention, the constructionof such engineered cells is achieved by introducing into a selected hostcell a recombinant DNA secretion construct in which DNA coding for asecretable form of the human GluR1B receptor i.e., a form of thereceptor bearing its native signal peptide or a functional, heterologousequivalent thereof, is linked operably with expression controllingelements that are functional in the selected host to drive expression ofthe receptor-encoding DNA, and thus elaborate the receptor protein inits desired, mature and membrane-bound form. Such cells are hereincharacterized as having the receptor-encoding DNA incorporated"expressibly" therein. The receptor-encoding DNA is referred to as"heterologous" with respect to the particular cellular host if such DNAis not naturally found in the particular host. The particular cell typeselected to serve as host for production of the human GluR1B receptorcan be any of several cell types currently available in the art, butshould not of course be a cell type that in its natural state elaboratesa surface receptor that can bind excitatory amino acids, and so confusethe assay results sought from the engineered cell line. Generally, suchproblems are avoided by selecting as host a non-neuronal cell type, andcan further be avoided using non-human cell lines, as is conventional.It will be appreciated that neuronal- and human-type cells maynevetheless serve as expression hosts, provided that "background"binding to the test ligand is accounted for in the assay results.

According to one embodiment of the present invention, the cell lineselected to serve as host for human GluR1B receptor production is amammalian cell. Several types of such cell lines are currently availablefor genetic engineering work, and these include the chinese hamsterovary (CHO) cells for example of K1 lineage (ATCC CCL 61) including thePro5 variant (ATCC CRL 1281); the fibroblast-like cells derived fromSV40-transformed African Green monkey kidney of the CV-1 lineage (ATCCCCL 70), of the COS-1 lineage (ATCC CRL 1650) and of the COS-7 lineage(ATCC CRL 1651); murine L-cells, murine 3T3 cells (ATCC CRL 1658),murine C127 cells, human embryonic kidney cells of the 293 lineage (ATCCCRL 1573), human carcinoma cells including those of the HeLa lineage(ATCC CCL 2), and neuroblastoma cells of the lines IMR-32 (ATCC CCL127), SK-N-MC (ATCC HTB 10) and SK-N-SH (ATCC HTB 11).

A variety of gene expression systems have been adapted for use withthese hosts and are now commercially available, and any one of thesesystems can be selected to drive expression of the receptor-encodingDNA. These systems, available typically in the form of plasmidicvectors, incorporate expression cassettes the functional components ofwhich include DNA constituting expression controlling sequences, whichare host-recognized and enable expression of the receptor-encoding DNAwhen linked 5' thereof. The systems further incorporate DNA sequenceswhich terminate expression when linked 3' of the receptor-encodingregion. Thus, for expression in the selected mammalian cell host, thereis generated a recombinant DNA expression construct in which DNA codingfor the receptor in secretable form is linked with expressioncontrolling DNA sequences recognized by the host, and which include aregion 5' of the receptor-encoding DNA to drive expression, and a 3'region to terminate expression. The plasmidic vector harbouring therecombinant DNA expression construct typically incorporates such otherfunctional components as an origin of replication, usuallyvirally-derived, to permit replication of the plasmid in the expressionhost and desirably also for plasmid amplification in a bacterial host,such as E. coli. To provide a marker enabling selection of stablytransformed recombinant cells, the vector will also incorporate a geneconferring some survival advantage on the transformants, such as a genecoding for neomycin resistance in which case the transformants areplated in medium supplemented with neomycin.

Included among the various recombinant DNA expression systems that canbe used to achieve mammalian cell expression of the receptor-encodingDNA are those that exploit promoters of viruses that infect mammaliancells, such as the promoter from the cytomegalovirus (CMV), the Roussarcoma virus (RSV), simian virus (SV40), murine mammary tumor virus(MMTV) and others. Also useful to drive expression are promoters such asthe LTR of retroviruses, insect cell promoters such as those regulatedby temperature, and isolated from Drosophila, as well as mammalian genepromoters such as those regulated by heavy metals i.e. themetalothionein gene promoter, and other steroid-inducible promoters.

For incorporation into the recombinant DNA expression vector, DNA codingfor the human GluR1B receptor, or an AMPA-binding fragment or variantthereof, can be obtained by applying selected techniques of geneisolation or gene synthesis. As described in more detail in the examplesherein, the human GluR1B receptor is encoded within the genome of humanbrain tissue, and can therefore be obtained from human DNA libraries bycareful application of conventional gene isolation and cloningtechniques. This typically will entail extraction of total messenger RNAfrom a fresh source of human brain tissue, preferably cerebellum orhippocampus tissue, followed by conversion of message to cDNA andformation of a library in for example a bacterial plasmid, moretypically a bacteriophage. Such bacteriophage harbouring fragments ofthe human DNA are typically grown by plating on a lawn of susceptible E.coli bacteria, such that individual phage plaques or colonies can beisolated. The DNA carried by the phage colony is then typicallyimmobilized on a nitrocellulose or nylon-based hybridization membrane,and then hybridized, under carefully controlled conditions, to aradioactively (or otherwise) labelled oligonucleotide probe ofappropriate sequence to identify the particular phage colony carryingreceptor-encoding DNA or fragment thereof, Typically, the gene or aportion thereof so identified is subcloned into a plasmidic vector fornucleic acid sequence analysis.

In a specific embodiment of the invention, the GluR1B receptor isencoded by the DNA sequence illustrated in FIG. 1 and SEQ ID NO:1. In anobvious alternative, the DNA sequences coding for the selected receptormay be a synonymous codon equivalent of the illustrated DNA sequences.

The illustrated DNA sequence constitutes the cDNA sequence identified inhuman brain cDNA libraries in the manner exemplified herein. Havingherein provided the nucleotide sequence of the human GluR1B receptor,however, it will be appreciated that polynucleotides encoding thereceptor can be obtained by other routes. Automated techniques of genesynthesis and/or amplification can be performed to generate DNA codingtherefor. Because of the length of the human GluR1B receptor-encodingDNA, application of automated synthesis may require staged geneconstruction, in which regions of the gene up to about 300 nucleotidesin length are synthesized individually and then ligated in correctsuccession by overhang complementarity for final assembly. Individuallysynthesized gene regions can be amplified prior to assembly, usingestablished polymerase chain reaction (PCR) technology.

The application of automated gene synthesis techniques provides anopportunity for generating polynucleotides that encode variants of thenaturally occurring human GluR1B receptor. It will be appreciated, forexample, that polynucleotides coding for the receptor can be generatedby substituting synonymous codons for those represented in the naturallyoccurring polynucleotide sequences herein identified. In addition,polynucleotides coding for human GluR1B receptor variants can begenerated which for example incorporate one or more, e.g. 1 to 10,single amino acid substitutions, deletions or additions. Since it willfor the most part be desirable to retain the natural ligand bindingprofile of the receptor for screening purposes, it is desirable to limitamino acid substitutions, for example to the so-called conservativereplacements in which amino acids of like charge are substituted, and tolimit substitutions to those sites less critical for receptor activitye.g. within about the first 20 N-terminal residues of the maturereceptor, and such other regions as are elucidated upon receptor domainmapping.

With appropriate template DNA in hand, the technique of PCRamplification may also be used to directly generate all or part of thefinal gene. In this case, primers are synthesized which will prime thePCR amplification of the final product, either in one piece, or inseveral pieces that may be ligated together. This may be via step-wiseligation of blunt ended, amplified DNA fragments, or preferentially viastep-wise ligation of fragments containing naturally occurringrestriction endonuclease sites. In this application, it is possible touse either cDNA or genomic DNA as the template for the PCRamplification. In the former case, the cDNA template can be obtainedfrom commercially available or self-constructed cDNA libraries ofvarious human brain tissues, including hippocampus and cerebellum.

Once obtained, the receptor-encoding DNA is incorporated for expressioninto any suitable expression vector, and host cells are transfectedtherewith using conventional procedures, such as DNA-mediatedtransformation, electroporation, or particle gun transformation.Expression vectors may be selected to provide transformed cell linesthat express the receptor-encoding DNA either transiently or in a stablemanner. For transient expression, host cells are typically transformedwith an expression vector harbouring an origin of replication functionalin a mammalian cell. For stable expression, such replication origins areunnecessary, but the vectors will typically harbour a gene coding for aproduct that confers on the transformants a survival advantage, toenable their selection. Genes coding for such selectable markers includethe E. coil gpt gene which confers resistance to mycophenolic acid, theneogene from transposon Tn5 which confers resistance to the antibioticG418 and to neomycin, the dhfr sequence from murine cells or E. coliwhich changes the phenotype of DHFR- cells into DHFR+ cells, and the tkgene of herpes simplex virus, which makes TK- cells phenotypically TK+cells. Both transient expression and stable expression can providetransformed cell lines, and membrane preparations derived therefrom, foruse in ligand screening assays.

For use in screening assays, cells transiently expressing thereceptor-encoding DNA can be stored frozen for later use, but becausethe rapid rate of plasmid replication will lead ultimately to celldeath, usually in a few days, the transformed cells should be used assoon as possible. Such assays may be performed either with intact cells,or with membrane preparations derived from such cells. The membranepreparations typically provide a more convenient substrate for theligand binding experiments, and are therefore preferred as bindingsubstrates. To prepare membrane preparations for screening purposes,i.e., ligand binding experiments, frozen intact cells are homogenizedwhile in cold water suspension and a membrane pellet is collected aftercentrifugation. The pellet is then washed in cold water, and dialyzed toremove endogenous EAA ligands such as glutamate, that would otherwisecompete for binding in the assays. The dialyzed membranes may then beused as such, or after storage in lyophilized form, in the ligandbinding assays. Alternatively, intact, fresh cells harvested about twodays after transient transfection or after about the same periodfollowing fresh plating of stably transfected cells, can be used forligand binding assays by the same methods as used for membranepreparations. When cells are used, the cells must be harvested by moregentle centrifugation so as not to damage them, and all washing must bedone in a buffered medium, for example in phosphate-buffered saline, toavoid osmotic shock and rupture of the cells.

The binding of a substance, i.e., a candidate ligand, to human GluR1Breceptor of the invention is evaluated typically using a predeterminedamount of cell-derived membrane (measured for example by proteindetermination), generally from about 25 ug to 100 ug. Generally,competitive binding assays will be useful to evaluate the affinity of atest compound relative to AMPA. This competitive binding assay can beperformed by incubating the membrane preparation with radiolabelledAMPA, for example [3H]-AMPA, in the presence of unlabelled test compoundadded at varying concentrations. Following incubation, either displacedor bound radiolabelled AMPA can be recovered and measured, to determinethe relative binding affinities of the test compound and AMPA for theparticular receptor used as substrate. In this way, the affinities ofvarious compounds for the AMPA-binding human CNS receptors can bemeasured. Alternatively, a radiolabelled analogue of glutamate may beemployed in place of radiolabelled AMPA, as competing ligand.

As an alternative to using cells that express receptor-encoding DNA,ligand characterization may also be performed using cells for exarnpleXenopus oocytes, that yield functional membrane-bound receptor followingintroduction by injection either of receptor-encoding messenger RNA intothe oocyte cytoplasm, or of receptor-encoding DNA into the oocytenucleus. To generate the messenger RNA of cytoplasmic delivery, thereceptor-encoding DNA is typically subcloned first into a plasmidicvector adjacent a suitable promoter region, such as the T3 or T7bacteriophage promoters, to enable transcription into RNA message. RNAis then transcribed from the inserted gene in vitro, collected and theninjected into Xenopus oocytes. Following the injection of nL volumes ofan RNA solution, the oocytes are left to incubate for up to severaldays, and are then tested for the ability to respond to a particularligand molecule supplied in a bathing solution. Since functional EAAreceptors act in part by operating a membrane channel through which ionsmay selectively pass, the functioning of the receptor in response to aparticular ligand molecule in the bathing solution may typically bemeasured as an electrical current utilizing microelectrodes insertedinto the cell, in the established manner.

In addition to using the receptor-encoding DNA to construct cell linesuseful for ligand screening, expression of the DNA can, according toanother aspect of the invention, be performed to produce fragments ofthe receptor in soluble form, for structure investigation, to raiseantibodies and for other experimental uses. It is expected that theportion of the human GluR1B receptor responsible for AMPA-bindingresides on the outside of the cell, i.e., is extracellular. It istherefore desirable in the first instance to facilitate thecharacterization of the receptor-ligand interaction by providing thisextracellular ligand-binding domain in quantity and in isolated form,i.e., free from the remainder of the receptor. To accomplish this, thefull-length human GluR receptor-encoding DNA may be modified bysite-directed mutagenesis, so as to introduce a translational stop codoninto the extracellular N-terminal region, immediately before thesequence encoding the first transmembrane domain (TM1), i.e., beforeresidue 521 as shown in FIG. 1 and SEQ ID NOs. 1 and 2. Since there willno longer be produced any transmembrane domain(s) to "anchor" thereceptor into the membrane, expression of the modified gene will resultin the secretion, in soluble form, of only the extracellularligand-binding domain. Standard ligand-binding assays may then beperformed to ascertain the degree of binding of a candidate compound tothe extracellular domain so produced. It may of course be necessary,using site-directed mutagenesis, to produce several different versionsof the extracellular regions, in order to optimize the degree of ligandbinding to the isolated domains.

Alternatively, it may be desirable to produce an extracellular domain ofthe receptor which is not derived from the amino-terminus of the matureprotein, but rather from the carboxy-terminus instead, for exampledomains immediately following the fourth transmembrane domain (TM4),i.e., residing between amino acid residues 809-888 inclusive (FIG. 1 andSEQ ID NOs. 1 and 2). In this case, site-directed mutagenesis and/orPCR-based amplification techniques may readily be used to provide adefined fragment of the gene encoding the receptor domain of interest.Such a DNA sequence may be used to direct the expression of the desiredreceptor fragment, either intracellularly, or in secreted fashion,provided that the DNA encoding the gene fragment is inserted adjacent toa translation start codon provided by the expression vector, and thatthe required translation reading frame is carefully conserved.

It will be appreciated that the production of such AMPA-bindingfragments of the human GluR1B receptor may be accomplished in a varietyof host cells. Mammalian cells such as CHO cells may be used for thispurpose, the expression typically being driven by an expression promotercapable of high-level expression, for example the CMV (cytomegalovirus)promoter. Alternately, non-mammalian cells, such as insect Sf9(Spodoptera frugiperda) cells may be used, with the expression typicallybeing driven by expression promoters of the baculovirus, for example thestrong, late polyhedrin protein promoter. Filamentous fungal expressionsystems may also be used to secrete large quantities of suchextracellular domains of the EAA receptor. Aspergillus nidulans, forexample, with the expression being driven by the alcA promoter, wouldconstitute such an acceptable system. In addition to such expressionhosts, it will be further appreciated that any prokaryotic or othereukaryotic expression system capable of expressing heterologous genes orgene fragments, whether intracellularly or extracellularly would besimilarly acceptable.

For use particularly in detecting the presence and/or location of ahuman GluR1B receptor, for example in brain tissue, the presentinvention also provides, in another of its aspects, labelled antibody tothe human GluR1B receptor. To raise such antibodies, there may be usedas immunogen either the intact, soluble receptor or an immunogenicfragment thereof i.e. a fragment capable of eliciting an immuneresponse, produced in a microbial or mammalian cell host as describedabove or by standard peptide synthesis techniques. Regions of humanGluR1B receptor particularly suitable for use as immunogenic fragmentsinclude those corresponding in sequence to an extracellular region ofthe receptor, or a portion of the extracellular region, such as peptidesconsisting of residues 1-520 or a fragment thereof comprising at leastabout 10 residues, including particularly fragments containing residues171-186 or 473-516; and peptides corresponding to the region betweentransmembrane domains TM-2 and TM-3, such as a peptide consisting ofresidues 586-595. Peptides consisting of the C-terminal domain (residues809-888), or fragment thereof, may also be used for the raising ofantibodies.

The raising of antibodies to the selected human GluR1B receptor orimmunogenic fragment can be achieved, for polyclonal antibodyproduction, using immunization protocols of conventional design, and anyof a variety of mammalian hosts, such as sheep, goats and rabbits.Alternatively, for monoclonal antibody production, immunocytes such assplenocytes can be recovered from the immunized animal and fused, usinghybridoma technology, to a myeloma cells. The fusion products are thenscreened by culturing in a selection medium, and cells producingantibody are recovered for continuous growth, and antibody recovery.Recovered antibody can then be coupled covalently to a detectable label,such as a radiolabel, enzyme label, luminescent label or the like, usinglinker technology established for this purpose.

In detectably labelled form, e.g. radiolabelled form, DNA or RNA codingfor a human GluR1B receptor, and selected regions thereof, may also beused, in accordance with another aspect of the present invention, ashybridization probes for example to identify sequence-related genesresident in the human or other mammalian genomes (or cDNA libraries) orto locate the human GluR1 B-encoding DNA in a specimen, such as braintissue. This can be done using either the intact coding region, or afragment thereof having radiolabelled e.g. 32p, nucleotides incorporatedtherein. To identify the human GluR1 B-encoding DNA in a specimen, it isdesirable to use either the full length cDNA coding therefor, or afragment which is unique thereto. With reference to FIG. 1, suchnucleotide fragments include those comprising at least about 17 nucleicacids, and otherwise corresponding in sequence to a region coding forthe extracellular N-terminal or C-terminal region of the receptor, orrepresenting a 5'-untranslated or 3'-untranslated region thereof. Sucholigonucleotide sequences, and the intact gene itself, may also be usedof course to clone human GluR1 B-related human genes, particularly cDNAequivalents thereof, by standard hybridization techniques.

EXAMPLE 1

Isolation of DNA coding for the human GluR1B receptor

cDNA coding for the human GluR1B receptor was identified by probinghuman fetal brain cDNA that was obtained as an EcoRI-based lambda phagelibrary (lambda ZAP) from Stratagene Cloning Systems (La Jolla, Calif.,U.S.A.). The cDNA library was screened using an oligonucleotide probecapable of annealing to the 5' region of the rat GluR1 receptor sequencereported by Hollmann et al, supra. The specific sequence of the32-P-labelled probe is provided below: (SEQ ID NOs:5)5'-CCAGATCGATATTGTGAACATCAGCGACACGTTTGAGATG-3'

The fetal brain cDNA library was screened under the followinghybridization conditions; 6×SSC, 25% formamide, 5% Dernhardt's solution,0.5% SDS, 100 ug/ml denatured salmon sperm DNA, 42C. Filters were washedwith 2×SSC containing 0.5% SDS at 25C for 5 minutes, followed by a 15minute wash at 50C with 2×SSC containing 0.5% SDS. The final wash waswith 1×SSC containing 0.5% SDS at 50C for 15 minutes. Filters wereexposed to X-ray film (Kodak) overnight. Of 10⁶ clones screened, onlyone cDNA insert, of about 3.2 kb, was identified, and designatedRKCSFG91, For sequencing, the '91 phage was plaque purified, thenexcised as a phagemid according to the supplier's specifications, togenerate an insert-carrying Bluescript-SK variant of the phagemidvector. Sequencing of the '91 clone across its entire sequence revealeda putative ATG initiation codon together with about 61 bases of5non-coding region and 2,718 bases of coding region. Also revealed was atermination codon, as well as about 438 bases of 3' non-translatedsequence. The entire sequence of the EcoRI/EcoRI insert is provided inFIG. 1 and SEQ ID NO:1.

A 6.2 kb phagemid designated pBS/humGluR1B, carrying thereceptor-encoding DNA as a 3.2 kb EcoRI/EcoRI insert in a 3.0 kbBluescript-SK phagemid background, was deposited, under the terms of theBudapest Treaty, with the American Type Culture Collection in Rockville,Md. U.S.A. on May 28, 1992, and has been assigned accession number ATCC75246.

EXAMPLE 2

Construction of genetically engineered cells producing human GluR1Breceptor

For transient expression in mammalian cells, cDNA coding for the humanGluR1B receptor was incorporated into the mammalian expression vectorpcDNA1, which is available commercially from Invitrogen Corporation (SanDiego, Calif., U.S.A.; catalogue number V490-20). This is amultifunctional 4.2 kb plasmid vector designed for cDNA expression ineukaryotic systems, and cDNA analysis in prokaryotes. Incorporated onthe vector are the CMV promoter and enhancer, splice segment andpolyadenylation signal, an SV40 and Polyoma virus origin of replication,and M13 origin to rescue single strand DNA for sequencing andmutagenesis, Sp6 and T7 RNA promoters for the production of sense andantisense RNA transcripts and a Col E1-like high copy plasmid origin. Apolylinker is located appropriately downstream of the CMV promoter (and3' of the T7 promoter).

The strategy depicted in FIG. 2 was employed to facilitate incorporationof the GluR1B receptor-encoding cDNA into an expression vector.Particularly, a Notl site was introduced onto the 3' flank of theBluescript-SK cDNA insert, and the cDNA insert was then released frompBS/humGluR1B as a 3.2 kb NotI/NotI fragment, which was thenincorporated at the NotI site in the pcDNAI polylinker. Sequencingacross the junctions was performed, to confirm proper insert orientationin pcDNA1. The resulting plasmid, designated pcDNA1/humGluR1B, was thenintroduced for transient expression into a selected mammalian cell host,in this case the monkey-derived, fibroblast like cells of the COS-1lineage (available from the American Type Culture Collection, Rockville,Md. as ATCC CRL 1650).

For transient expression of the GluR1B-encoding DNA, COS-1 cells weretransfected with approximately 8 ug DNA (as pcDNA1/humGluR2B) per 10⁶COS cells, by DEAE-mediated DNA transfection and treated withchloroquine according to the procedures described by Maniatis et al,supra. Briefly, COS-1 cells were plated at a density of 5×10⁶ cells/dishand then grown for 24 hours in FBS-supplemented DMEM/F12 medium. Mediumwas then removed and cells were washed in PBS and then in medium. Therewas then applied on the cells 10 ml of a transfection solutioncontaining DEAE dextran (0.4 mg/ml), 100 uM chloroquine, 10% NuSerum,DNA (0.4 mg/ml)in DMEM/F12 medium. After incubation for 3 hours at 37C,cells were washed in PBS and medium as just described and then shockedfor 1 minute with 10% DMSO in DMEM/F12 medium. Cells were allowed togrow for 2-3 days in 10% FBS-supplemented medium, and at the end ofincubation dishes were placed on ice, washed with ice cold PBS and thenremoved by scraping. Cells were then harvested by centrifugation at 1000rpm for 10 minutes and the cellular pellet was frozen in liquidnitrogen, for subsequent use in ligand binding assays. Northern blotanalysis of a thawed aliquot of frozen cells confirmed expression ofreceptor-encoding cDNA in cells under storage.

In a like manner, stably transfected cell lines can also prepared usingtwo different cell types as host: CHO K1 and CHO Pro5. To constructthese cell lines, cDNA coding for human GluR1B is incorporated into themammalian expression vector pRC/CMV (Invitrogen), which enables stableexpression. Insertion at this site placed the cDNA under the expressioncontrol of the cytomegalovirus promoter and upstream of thepolyadenylation site and terminator of the bovine growth hormone gene,and into a vector background comprising the neomycin resistance gene(driven by the SV40 early promoter) as selectable marker.

To introduce plasmids constructed as described above, the host CHO cellsare first seeded at a density of 5×10⁵ in 10% FBS-supplemented MEMmedium. After growth for 24 hours, fresh medium are added to the platesand three hours later, the cells are transfected using the calciumphosphate-DNA co-precipitation procedure (Maniatis et al, supra).Briefly, 3 ug of DNA is mixed and incubated with buffered calciumsolution for 10 minutes at room temperature. An equal volume of bufferedphosphate solution is added and the suspension is incubated for 15minutes at room temperature. Next, the incubated suspension is appliedto the cells for 4 hours, removed and cells were shocked with mediumcontaining 15% glycerol. Three minutes later, cells are washed withmedium and incubated for 24 hours at normal growth conditions. Cellsresistant to neomycin are selected in 10% FBS-supplemented alpha-MEMmedium containing G418 (1 mg/ml). Individual colonies of G418-resistantcells are isolated about 2-3 weeks later, clonally selected and thenpropogated for assay purposes.

EXAMPLE 3

Ligand binding assays

Transfected cells in the frozen state were resuspended in ice-colddistilled water using a hand homogenizer, sonicated for 5 seconds, andthen centrifuged for 20 minutes at 50,000 g. The supernatant wasdiscarded and the membrane pellet stored frozen at -70° C.

COS cell membrane pellets were suspended in ice cold 50 mM Tris-HCl (pH7.55, 5C) and centrifuged again at 50,000 g for 10 minutes in order toremove endogenous glutamate that would compete for binding. Pellets wereresuspended in ice cold 50 mM Tris-HCl (pH 7.55) buffer and theresultant membrane preparation was used as tissue source for bindingexperiments described below. Proteins were determined using the PierceReagent with BSA as standard.

Binding assays were then performed, using an amount of COS-derivedmembrane equivalent to from 25-100 ug as judged by protein determinationand selected radiolabelled ligand. In particular, for AMPA-bindingassays, incubation mixtures consisted of 25-100 ug tissue protein andD,L-alpha-[5-methyl-3H]amino-3-hydroxy-5-methylisoxazole-4-propionicacid (3H-AMPA, 27.6Ci/mmole, 10 nM final) with 0.1M KSCN and 2.5 mMCaCl₂ in the 1 ml final volume. Non-specific binding was determined inthe presence of 1 mM L-glutamate. Samples were incubated on ice for 60minutes in plastic minivials, and bound and free ligand were separatedby centrifugation for 30 minutes at 50,000 g. Pellets were washed twicein 4 ml of the cold incubation buffer, then 5 ml of BeckmanReady-Protein Plus scintillation cocktail was added, for counting.

For kainate-binding assays, incubation mixtures consisted of 25-100 ugtissue protein and [vinylidene-3H] kainic acid (58Ci/mmole, 5 nM final)in the cold incubation buffer, 1 ml final volume. Non-specific bindingwas determined in the presence of 1 mM L-glutamate. Samples wereincubated as for the AMPA-binding assays, and bound and free ligand wereseparated by rapid filtration using a Brandel cell harvester and GF/Bfilters pre-soaked in ice-cold 0.3% polyethyleneimine. Filters werewashed twice in 6 ml of the cold incubation buffer, then placed inscintillation vials with 5 ml of Beckman Ready-Protein Plusscintillation cocktail for counting.

Assays performed in this manner, using membrane preparations derivedfrom the human GluR1B receptor-producing COS cells, revealed specificbinding of about 100-150 fmole/mg protein, at 10 nM [3H]-AMPA (FIG. 3).Mock transfected cells exhibited no specific binding of any of theligands tested. These results demonstrate clearly that the human GluR1Breceptor is binding AMPA with specificity. This activity, coupled withthe fact that there is little or no demonstrable binding of eitherkainate or NMDA, clearly assigns the human GluR1B receptor to be of theAMPA type of EAA receptor. Furthermore, this binding profile indicatesthat the receptor is binding in an authentic manner, and can thereforereliably predict the ligand binding "signature" of its non-recombinantcounterpart from the human brain. These features make the recombinantreceptor especially useful for selecting and characterizing ligandcompounds which bind to the receptor, and/or for selecting andcharacterizing compounds which may act by displacing other ligands fromthe receptor. The isolation of the GluR1B receptor genes insubstantially pure form, capable of being expressed as a single,homogeneous receptor species, therefore frees the ligand binding assayfrom the lack of precision introduced when complex, heterogeneousreceptor preparations from human and other mammalian brains are used toattempt such characterizations.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3220 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 62..2782                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 62..115                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 116..2782                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCCACACCAAATCTATGATTGGACCTGGGCTTCTTTTTCGCCAATGCAAAAAGGAA60                TATGCAGCACATTTTTGCCTTCTTCTGCACCGGTTTCCTAGGCGCG106                             MetGlnHisIlePheAlaPhePheCysThrGlyPheLeuGlyAla                                 18-15- 10-5                                                                   GTAGTAGGTGCCAATTTCCCCAACAATATCCAGATCGGGGGATTATTT154                           ValValGlyAlaAsnPheProAsnAsnIleGlnIleGlyGlyLeuPhe                              1510                                                                          CCAAACCAGCAGTCACAGGAACATGCTGCTTTTAGATTTGCTTTGTCG202                           ProAsnGlnGlnSerGlnGluHisAlaAlaPheArgPheAlaLeuSer                              152025                                                                        CAACTCACAGAGCCCCCGAAGCTGCTCCCCCAGATTGATATTGTGAAC250                           GlnLeuThrGluProProLysLeuLeuProGlnIleAspIleValAsn                              30354045                                                                      ATCAGCGACACGTTTGAGATGACCTATAGATTCTGTTCCCAGTTCTCC298                           IleSerAspThrPheGluMetThrTyrArgPheCysSerGlnPheSer                              505560                                                                        AAAGGAGTCTATGCCATCTTTGGGTTTTATGAACGTAGGACTGTCAAC346                           LysGlyValTyrAlaIlePheGlyPheTyrGluArgArgThrValAsn                              657075                                                                        ATGCTGACCTCCTTTTGTGGGGCCCTCCACGTCTGCTTCATTACGCCG394                           MetLeuThrSerPheCysGlyAlaLeuHisValCysPheIleThrPro                              808590                                                                        AGCTTTCCCGTTGATACATCCAATCAGTTTGTCCTTCAGCTGCGCCCT442                           SerPheProValAspThrSerAsnGlnPheValLeuGlnLeuArgPro                              95100105                                                                      GAACTGCAGGATGCCCTCATCAGCATCATTGACCATTACAAGTGGCAG490                           GluLeuGlnAspAlaLeuIleSerIleIleAspHisTyrLysTrpGln                              110115120125                                                                  AAATTTGTCTACATTTATGATGCCGACCGGGGCTTATCCGTCCTGCAG538                           LysPheValTyrIleTyrAspAlaAspArgGlyLeuSerValLeuGln                              130135140                                                                     AAAGTCCTGGATACAGCTGCTGAGAAGAACTGGCAGGTGACAGCAGTC586                           LysValLeuAspThrAlaAlaGluLysAsnTrpGlnValThrAlaVal                              145150155                                                                     AACATTTTGACAACCACAGAGGAGGGATACCGGATGCTCTTTCAGGAC634                           AsnIleLeuThrThrThrGluGluGlyTyrArgMetLeuPheGlnAsp                              160165170                                                                     CTGGAGAAGAAAAAGGAGCGGCTGGTGGTGGTGGACTGTGAATCAGAA682                           LeuGluLysLysLysGluArgLeuValValValAspCysGluSerGlu                              175180185                                                                     CGCCTCAATGCTATCTTGGGCCAGATTATAAAGCTAGAGAAGAATGGC730                           ArgLeuAsnAlaIleLeuGlyGlnIleIleLysLeuGluLysAsnGly                              190195200205                                                                  ATCGGCTACCACTACATTCTTGCAAATCTGGGCTTCATGGACATTGAC778                           IleGlyTyrHisTyrIleLeuAlaAsnLeuGlyPheMetAspIleAsp                              210215220                                                                     TTAAACAAATTCAAGGAGAGTGGCGCCAATGTGACAGGTTTCCAGCTG826                           LeuAsnLysPheLysGluSerGlyAlaAsnValThrGlyPheGlnLeu                              225230235                                                                     GTGAACTACACAGACACTATTCCGGCCAAGATCATGCAGCAGTGGAAG874                           ValAsnTyrThrAspThrIleProAlaLysIleMetGlnGlnTrpLys                              240245250                                                                     AATAGTGATGCTCGAGACCACACACGGGTGGACTGGAAGAGACCCAAG922                           AsnSerAspAlaArgAspHisThrArgValAspTrpLysArgProLys                              255260265                                                                     TACACCTCTGCGCTCACCTACGATGGGGTGAAGGTGATGGCTGAGGCT970                           TyrThrSerAlaLeuThrTyrAspGlyValLysValMetAlaGluAla                              270275280285                                                                  TTCCAGAGCCTGCGGAGGCAGAGAATTGATATATCTCGCCGGGGGAAT1018                          PheGlnSerLeuArgArgGlnArgIleAspIleSerArgArgGlyAsn                              290295300                                                                     GCTGGGGATTGTCTGGCTAACCCAGCTGTTCCCTGGGGCCAAGGGATC1066                          AlaGlyAspCysLeuAlaAsnProAlaValProTrpGlyGlnGlyIle                              305310315                                                                     GACATCCAGAGAGCTCTGCAGCAGGTGCGATTTGAAGGTTTAACAGGA1114                          AspIleGlnArgAlaLeuGlnGlnValArgPheGluGlyLeuThrGly                              320325330                                                                     AACGTGCAGTTTAATGAGAAAGGACGCCGGACCAACTACACGCTCCAC1162                          AsnValGlnPheAsnGluLysGlyArgArgThrAsnTyrThrLeuHis                              335340345                                                                     GTGATTGAAATGAAACATGACGGCATCCGAAAGATTGGTTACTGGAAT1210                          ValIleGluMetLysHisAspGlyIleArgLysIleGlyTyrTrpAsn                              350355360365                                                                  GAAGATGATAAGTTTGTCCCTGCAGCCACCGATGCCCAAGCTGGGGGC1258                          GluAspAspLysPheValProAlaAlaThrAspAlaGlnAlaGlyGly                              370375380                                                                     GATAATTCAAGTGTTCAGAACAGAACATACATCGTCACAACAATCCTA1306                          AspAsnSerSerValGlnAsnArgThrTyrIleValThrThrIleLeu                              385390395                                                                     GAAGATCCTTATGTGATGCTCAAGAAGAACGCCAATCAGTTTGAGGGC1354                          GluAspProTyrValMetLeuLysLysAsnAlaAsnGlnPheGluGly                              400405410                                                                     AATGACCGTTACGAGGGCTACTGTGTAGAGCTGGCGGCAGAGATTGCC1402                          AsnAspArgTyrGluGlyTyrCysValGluLeuAlaAlaGluIleAla                              415420425                                                                     AAGCACGTGGGCTACTCCTACCGTCTGGAGATTGTCAGTGATGGAAAA1450                          LysHisValGlyTyrSerTyrArgLeuGluIleValSerAspGlyLys                              430435440445                                                                  TACGGAGCCCGAGACCCTGACACGAAGGCCTGGAATGGCATGGTGGGA1498                          TyrGlyAlaArgAspProAspThrLysAlaTrpAsnGlyMetValGly                              450455460                                                                     GAGCTGGTCTATGGAAGAGCAGATGTGGCTGTGGCTCCCTTAACTATC1546                          GluLeuValTyrGlyArgAlaAspValAlaValAlaProLeuThrIle                              465470475                                                                     ACTTTGGTCCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATGAGT1594                          ThrLeuValArgGluGluValIleAspPheSerLysProPheMetSer                              480485490                                                                     TTGGGGATCTCCATCATGATTAAAAAACCACAGAAATCCAAGCCGGGT1642                          LeuGlyIleSerIleMetIleLysLysProGlnLysSerLysProGly                              495500505                                                                     GTCTTCTCCTTCCTTGATCCTTTGGCTTATGAGATTTGGATGTGCATT1690                          ValPheSerPheLeuAspProLeuAlaTyrGluIleTrpMetCysIle                              510515520525                                                                  GTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGC1738                          ValPheAlaTyrIleGlyValSerValValLeuPheLeuValSerArg                              530535540                                                                     TTCAGTCCCTATGAATGGCACAGTGAAGAGTTTGAGGAAGGACGGGAC1786                          PheSerProTyrGluTrpHisSerGluGluPheGluGluGlyArgAsp                              545550555                                                                     CAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTTG1834                          GlnThrThrSerAspGlnSerAsnGluPheGlyIlePheAsnSerLeu                              560565570                                                                     TGGTTCTCCCTGGGAGCCTTCATGCAGCAAGGATGTGACATTTCTCCC1882                          TrpPheSerLeuGlyAlaPheMetGlnGlnGlyCysAspIleSerPro                              575580585                                                                     AGGTCCCTGTCTGGTCGCATCGTTGGTGGCGTCTGGTGGTTCTTCACC1930                          ArgSerLeuSerGlyArgIleValGlyGlyValTrpTrpPhePheThr                              590595600605                                                                  TTAATCATCATCTCCTCATATACAGCCAATCTGGCCGCCTTCCTGACC1978                          LeuIleIleIleSerSerTyrThrAlaAsnLeuAlaAlaPheLeuThr                              610615620                                                                     GTGGAGAGGATGGTGTCTCCCATTGAGAGTGCAGAGGACCTAGCGAAC2026                          ValGluArgMetValSerProIleGluSerAlaGluAspLeuAlaAsn                              625630635                                                                     GAGACAGAAATTGCCTACGGGACGCTGGAAGCAGGATCTACTAAGGAG2074                          GluThrGluIleAlaTyrGlyThrLeuGluAlaGlySerThrLysGlu                              640645650                                                                     TTCTTCAGGAGGTCTAAAATTGCTGTGTTTGAGAAGATGTGGACATAC2122                          PhePheArgArgSerLysIleAlaValPheGluLysMetTrpThrTyr                              655660665                                                                     ATGAAGTCAGCAGAGCCATCAGTTTTTGTGCGGACCACAGAGGAGGGG2170                          MetLysSerAlaGluProSerValPheValArgThrThrGluGluGly                              670675680685                                                                  ATGATTCGAGTGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAG2218                          MetIleArgValArgLysSerLysGlyLysTyrAlaTyrLeuLeuGlu                              690695700                                                                     TCCACCATGAATGAGTACATTGAGCAGCGGAAACCCTGTGACACCATG2266                          SerThrMetAsnGluTyrIleGluGlnArgLysProCysAspThrMet                              705710715                                                                     AAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCC2314                          LysValGlyGlyAsnLeuAspSerLysGlyTyrGlyIleAlaThrPro                              720725730                                                                     AAGGGGTCTGCCCTGAGAGGTCCCGTAAACCTAGCGGTTTTGAAACTC2362                          LysGlySerAlaLeuArgGlyProValAsnLeuAlaValLeuLysLeu                              735740745                                                                     AGTGAGCAAGGCGTCTTAGACAAGCTGAAAAGCAAATGGTGGTACGAT2410                          SerGluGlnGlyValLeuAspLysLeuLysSerLysTrpTrpTyrAsp                              750755760765                                                                  AAAGGGGAATGTGGAAGCAAGGACTCCGGAAGTAAGGACAAGACAAGC2458                          LysGlyGluCysGlySerLysAspSerGlySerLysAspLysThrSer                              770775780                                                                     GCTCTGAGCCTCAGCAATGTGGCAGGCGTGTTCTACATCCTGATCGGA2506                          AlaLeuSerLeuSerAsnValAlaGlyValPheTyrIleLeuIleGly                              785790795                                                                     GGACTTGGACTAGCCATGCTGGTTGCCTTAATCGAGTTCTGCTACAAA2554                          GlyLeuGlyLeuAlaMetLeuValAlaLeuIleGluPheCysTyrLys                              800805810                                                                     TCCCGTAGTGAATCCAAGCGGATGAAGGGTTTTTGTTTGATCCCACAG2602                          SerArgSerGluSerLysArgMetLysGlyPheCysLeuIleProGln                              815820825                                                                     CAATCCATCAACGAAGCCATACGGACATCGACCCTCCCCCGCAACAGC2650                          GlnSerIleAsnGluAlaIleArgThrSerThrLeuProArgAsnSer                              830835840845                                                                  GGGGCAGGAGCCAGCAGCGGCGGCAGTGGAGAGAATGGTCGGGTGGTC2698                          GlyAlaGlyAlaSerSerGlyGlySerGlyGluAsnGlyArgValVal                              850855860                                                                     AGCCATGACTTCCCCAAGTCCATGCAATCGATTCCTTGCATGAGCCAC2746                          SerHisAspPheProLysSerMetGlnSerIleProCysMetSerHis                              865870875                                                                     AGTTCAGGGATGCCCTTGGGAGCCACGGGATTGTAACTGGAGCAGATGGAGAC2799                     SerSerGlyMetProLeuGlyAlaThrGlyLeu                                             880885                                                                        CCCTTGGGGAGCAGGCTCGGGCTCCCCAGCCCCATCCCAAACCCTTCAGTGCCAAAAACA2859              ACAACAAAATAGAAAGCGCAACCACCACCAACCACTGCGACCACAAGAAGGATGATTCAA2919              CAGGTTTTCCTGAAGAATTGAAAAACCATTTTGCTGTCCCTTTTCCTTTTTTGATGTTCT2979              TTCACCCTTTTCTGTTTGCTAAGTGAGGATGAAAAAATAACACTGTACTGCAATAAGGGG3039              AGAGTAACCCTGTCTAATGAAACCTGTGTCTCTGAGAGTAGAGTCACTGGAACACTAATG3099              AGGAAACTGCACTGTTTTATTTTAATTCAGTTGTTAGTGTGTCTTAGTGTGTGCAATTTT3159              TTTTCTTACTAATATCCATGGTTTGCAGGTTCTGTTAGGCCCTTTCCTTCTCCTGGAATT3219              C3220                                                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 906 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlnHisIlePheAlaPhePheCysThrGlyPheLeuGlyAlaVal                              18-15-10-5                                                                    ValGlyAlaAsnPheProAsnAsnIleGlnIleGlyGlyLeuPhePro                              1510                                                                          AsnGlnGlnSerGlnGluHisAlaAlaPheArgPheAlaLeuSerGln                              15202530                                                                      LeuThrGluProProLysLeuLeuProGlnIleAspIleValAsnIle                              354045                                                                        SerAspThrPheGluMetThrTyrArgPheCysSerGlnPheSerLys                              505560                                                                        GlyValTyrAlaIlePheGlyPheTyrGluArgArgThrValAsnMet                              657075                                                                        LeuThrSerPheCysGlyAlaLeuHisValCysPheIleThrProSer                              808590                                                                        PheProValAspThrSerAsnGlnPheValLeuGlnLeuArgProGlu                              95100105110                                                                   LeuGlnAspAlaLeuIleSerIleIleAspHisTyrLysTrpGlnLys                              115120125                                                                     PheValTyrIleTyrAspAlaAspArgGlyLeuSerValLeuGlnLys                              130135140                                                                     ValLeuAspThrAlaAlaGluLysAsnTrpGlnValThrAlaValAsn                              145150155                                                                     IleLeuThrThrThrGluGluGlyTyrArgMetLeuPheGlnAspLeu                              160165170                                                                     GluLysLysLysGluArgLeuValValValAspCysGluSerGluArg                              175180185190                                                                  LeuAsnAlaIleLeuGlyGlnIleIleLysLeuGluLysAsnGlyIle                              195200205                                                                     GlyTyrHisTyrIleLeuAlaAsnLeuGlyPheMetAspIleAspLeu                              210215220                                                                     AsnLysPheLysGluSerGlyAlaAsnValThrGlyPheGlnLeuVal                              225230235                                                                     AsnTyrThrAspThrIleProAlaLysIleMetGlnGlnTrpLysAsn                              240245250                                                                     SerAspAlaArgAspHisThrArgValAspTrpLysArgProLysTyr                              255260265270                                                                  ThrSerAlaLeuThrTyrAspGlyValLysValMetAlaGluAlaPhe                              275280285                                                                     GlnSerLeuArgArgGlnArgIleAspIleSerArgArgGlyAsnAla                              290295300                                                                     GlyAspCysLeuAlaAsnProAlaValProTrpGlyGlnGlyIleAsp                              305310315                                                                     IleGlnArgAlaLeuGlnGlnValArgPheGluGlyLeuThrGlyAsn                              320325330                                                                     ValGlnPheAsnGluLysGlyArgArgThrAsnTyrThrLeuHisVal                              335340345350                                                                  IleGluMetLysHisAspGlyIleArgLysIleGlyTyrTrpAsnGlu                              355360365                                                                     AspAspLysPheValProAlaAlaThrAspAlaGlnAlaGlyGlyAsp                              370375380                                                                     AsnSerSerValGlnAsnArgThrTyrIleValThrThrIleLeuGlu                              385390395                                                                     AspProTyrValMetLeuLysLysAsnAlaAsnGlnPheGluGlyAsn                              400405410                                                                     AspArgTyrGluGlyTyrCysValGluLeuAlaAlaGluIleAlaLys                              415420425430                                                                  HisValGlyTyrSerTyrArgLeuGluIleValSerAspGlyLysTyr                              435440445                                                                     GlyAlaArgAspProAspThrLysAlaTrpAsnGlyMetValGlyGlu                              450455460                                                                     LeuValTyrGlyArgAlaAspValAlaValAlaProLeuThrIleThr                              465470475                                                                     LeuValArgGluGluValIleAspPheSerLysProPheMetSerLeu                              480485490                                                                     GlyIleSerIleMetIleLysLysProGlnLysSerLysProGlyVal                              495500505510                                                                  PheSerPheLeuAspProLeuAlaTyrGluIleTrpMetCysIleVal                              515520525                                                                     PheAlaTyrIleGlyValSerValValLeuPheLeuValSerArgPhe                              530535540                                                                     SerProTyrGluTrpHisSerGluGluPheGluGluGlyArgAspGln                              545550555                                                                     ThrThrSerAspGlnSerAsnGluPheGlyIlePheAsnSerLeuTrp                              560565570                                                                     PheSerLeuGlyAlaPheMetGlnGlnGlyCysAspIleSerProArg                              575580585590                                                                  SerLeuSerGlyArgIleValGlyGlyValTrpTrpPhePheThrLeu                              595600605                                                                     IleIleIleSerSerTyrThrAlaAsnLeuAlaAlaPheLeuThrVal                              610615620                                                                     GluArgMetValSerProIleGluSerAlaGluAspLeuAlaAsnGlu                              625630635                                                                     ThrGluIleAlaTyrGlyThrLeuGluAlaGlySerThrLysGluPhe                              640645650                                                                     PheArgArgSerLysIleAlaValPheGluLysMetTrpThrTyrMet                              655660665670                                                                  LysSerAlaGluProSerValPheValArgThrThrGluGluGlyMet                              675680685                                                                     IleArgValArgLysSerLysGlyLysTyrAlaTyrLeuLeuGluSer                              690695700                                                                     ThrMetAsnGluTyrIleGluGlnArgLysProCysAspThrMetLys                              705710715                                                                     ValGlyGlyAsnLeuAspSerLysGlyTyrGlyIleAlaThrProLys                              720725730                                                                     GlySerAlaLeuArgGlyProValAsnLeuAlaValLeuLysLeuSer                              735740745750                                                                  GluGlnGlyValLeuAspLysLeuLysSerLysTrpTrpTyrAspLys                              755760765                                                                     GlyGluCysGlySerLysAspSerGlySerLysAspLysThrSerAla                              770775780                                                                     LeuSerLeuSerAsnValAlaGlyValPheTyrIleLeuIleGlyGly                              785790795                                                                     LeuGlyLeuAlaMetLeuValAlaLeuIleGluPheCysTyrLysSer                              800805810                                                                     ArgSerGluSerLysArgMetLysGlyPheCysLeuIleProGlnGln                              815820825830                                                                  SerIleAsnGluAlaIleArgThrSerThrLeuProArgAsnSerGly                              835840845                                                                     AlaGlyAlaSerSerGlyGlySerGlyGluAsnGlyArgValValSer                              850855860                                                                     HisAspPheProLysSerMetGlnSerIleProCysMetSerHisSer                              865870875                                                                     SerGlyMetProLeuGlyAlaThrGlyLeu                                                880885                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGCTTGCGGCCGC13                                                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCGGCCGCA9                                                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic DNA oligonucleotide                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCAGATCGATATTGTGAACATCAGCGACACGTTTGAGATG40                                    __________________________________________________________________________

We claim:
 1. An isolated polynucleotide which encodes a protein having the sequence of SEQ ID NO:2.
 2. An isolated polynucleotide according to claim 1, which consists of DNA.
 3. An isolated polynucleotide according to claim 1, which consists of RNA.
 4. A recombinant DNA vector having incorporated therein a polynucleotide which encodes a protein having the sequence of SEQ ID NO:2.
 5. A recombinant DNA vector according to claim 4, wherein the polynucleotide incorporated therein is linked operably with DNA enabling expression and secretion of said receptor in a cellular host.
 6. A recombinant DNA vector according to claim 5, which is the plasmid pBS/humGluR1B (ATCC 75246).
 7. A 3.2 kilobase, EcoRI/EcoRI fragment of the recombinant DNA construct defined in claim
 6. 8. A cellular host having incorporated therein a heterologous polynucleotide which encodes a protein having the sequence of SEQ ID NO:2.
 9. A cellular host according to claim 8, which is a mammalian cell.
 10. A membrane preparation derived from a cellular host as defined in claim
 9. 11. A cellular host according to claim 8, which is an oocyte.
 12. A membrane preparation derived from a cellular host as defined in claim
 8. 13. A process for obtaining a substantially homogeneous source of a human GluR1B receptor, which comprises the steps of culturing a cellular host having incorporated expressibly therein a polynucleotide that encodes a protein having the sequence of SEQ ID NO:2, and then recovering the cultured cells.
 14. A process for obtaining a substantially homogeneous source of a human GluR1B receptor according to claim 13, comprising the subsequent step of obtaining a membrane preparation from the cultured cells. 